The regulated release of neurotransmitter by exocytosis depends on transport of the transmitter into neurosecretory vesicles. We previously found that several so-called type I phosphate transporters in fact transport glutamate into synaptic vesicles, providing the first unambiguous markers for neurons that use glutamate as a transmitter. Vesicular glutamate transporters (VGLUTs) 1 and 2 exhibit an almost mutually exclusive pattern of expression in the adult brain, with VGLUT1 in cortex and hippocampus, and VGLUT2 in the thalamus and brainstem. The isoforms do not apparently differ in intrinsic transport activity, but the analysis of VGLUT1 knock-out mice suggests differences in response of the two isoforms to high frequency stimulation, presumably due to differences in membrane trafficking. We have now found that VGLUT1 contains multiple internalization motifs, only one of which is shared with VGLUT2, and we have begun to identify the proteins that interact with these sequences. In addition, the VGLUT1 knock-out has suggested a transient, developmental role for VGLUT2 in cortical and hippocampal neurons destined to make only VGLUT1 in adulthood. In contrast to VGLUT1 and 2, VGLUTS is expressed by a number of cell populations not traditionally considered to release glutamate, including cholinergic interneurons of the striatum, GABAergic interneurons in the cortex and hippocampus, serotonin and perhaps dopamine neurons. The long-term objectives of this program are to understand how the release of glutamate influences neural development, synaptic transmission and behavior. The strategy is to study the function and trafficking of VGLUTs using biophysical methods, and to determine their role in vivo by genetic manipulation in mice. In terms of specific aims, we will: 1) Characterize the protein interactions of VGLUT1. To understand how protein interactions influence the trafficking of VGLUT1, we will use optical imaging in live neurons. 2) Determine the role of transient VGLUT2 expression during development. We will use conditional VGLUT2 knock-out mice to determine the effects of early VGLUT2 expression on axon guidance, synapse development, function and plasticity. 3) Identify the roles of glutamate release by cell populations expressing VGLUTS. We will use VGLUTS knock-out and BAG transgenic mice that label VGLUT3+ neurons with GFP to assess the physiological and behavioral roles of VGLUTS expression by diverse neural systems. We will also produce a conditional knock-out to distinguish among the behavioral effects of VGLUTS expressed by different neurons.